Identifizierung von physiologischen und artifiziellen Liganden von GABARAP und Charakterisierung der resultierenden Interaktionen

Mohrlüder, Jeannine

Dissertation / PhD Thesis/Book

Identifizierung von physiologischen und artifiziellen Liganden von GABARAP und Charakterisierung der resultierenden Interaktionen

Mohrlüder, J. (Corresponding author)FZJ*

2008
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-89336-511-1

Jülich : Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag, Schriften des Forschungszentrums Jülich. Reihe Gesundheit / Health 3, (2008) = Universität Düsseldorf, Diss., 2007

Please use a persistent id in citations: http://hdl.handle.net/2128/3120

Abstract: $\gamma$-aminobutyric acid (GABA) receptors (type A) (GABA$_{A}$ receptors) mediate fast synaptic inhibition in the central nervous system. They are of particular pharmacological importance and are targets for drugs used to treat mental disorders or to modulate sleep and mood. Sorting, transport and degradation of neurotransmitter receptors are important for the construction and maintenance of functional synapses and are fundamental processes to modulate synaptic plasticity. The GABA$_{A}$ receptor-associated protein (GABARAP) belongs to the MAP-LC3 protein family which is involved in vesicular transport processes, like autophagocytosis and intra-golgi transport. GABARAP binds to a GABA$_{A}$ receptor subunit and participates in its transport events. The exact mechanisms and the function of this interaction are not yet known. The objective of the present work was the i dentificati on of artificial peptide and physiological protein ligands of GABARAP and the characterisation of the resulting interactions. This enhances on the one hand the understanding of the binding mechanisms of any ligand to GABARAP and on the other hand, can facilitate the design of new drugs capable of modulating the function of GABARAP or the GABA$_{A}$ receptor. The knowledge of new GABARAP interaction partners additionally contributes to a better understanding of the cellular functions of GABARAP and the procedures at the postsynaptic membrane. By employing a phage display selection procedure, artificial high-affinity peptide ligands of GABARAP could successfully be identified. The resulting interactions could be characterised via enzyme-linked immunosorbent assay, fluorescence titration, surface-plasmon resonance and nuclear magnetic resonance spectroscopy. A consensus motif was derived from the GABARAP-binding peptide sequences. It was used to search protein databases to identify putative GABARAP-binding proteins. For the first time, the human cellular proteins calreticulin (CRT) and clathrin heavy chain (CHC) were identified to bind GABARAP. The corresponding interactions could be characterised via surface plasmon resonance, pulldown analysis and nuclear magnetic resonance spectroscopy. The results of the present work make a substantial contribution to an enhanced understanding of the GABARAP binding specificity and its cellular function.

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